Osteopontin (referred to as “OPN” hereinbelow) is an acidic, calcium-binding glycoprotein abundant in bone. It has been known that three types of human OPN isoforms namely osteopontin-a (referred to as “OPN-a” hereinbelow), osteopontin-b (referred to as “OPN-b” hereinbelow) and osteopontin-c (referred to as “OPN-c” hereinbelow) are naturally generated by alternative splicing (Y. Saitoh et al., (1995): Laboratory Investigation, 72, 55–63). It has been believed that among them, the precursor of OPN-a has an amino acid sequence shown below as SEQ ID NO. 1 in the Sequence Listing, where the signal peptide is cleaved on secretion, so that the mature form OPN-a of I17-N314 is prepared. Additionally, the mature OPN is cleaved at the C-terminal side of the 168-th residue arginine with thrombin in a biological organism into two fragments, namely N-terminal and C-terminal fragments.
OPN described above has various physiologically, pathologically significant functions, for example cell adhesion, cell migration, tumorigenesis, immune response and inhibition of complement-mediated cytolysis. Various types of receptors on cellular surface mediate the various functions. OPN has the RGD sequence therein (for example, OPN-a has the sequence from the residue at position 159 to the residue at position 161). Integrin species recognizing the RGD sequence such as αVβ3, αVβ1 and αVβ5 are major OPN receptors; specifically, the integrin species αVβ3, αVβ1 and αVβ5 mediate cell adhesion in vascular smooth muscle cells. Further, αVβ3 is involved in the migrations of macrophages, lymphocytes, endothelial cells, and smooth muscle cells and the like.
Further, research works so far have elucidated that OPN also binds through the sequence SVVYGLR (residues 11–17 of SEQ ID NO: 1) to α9β1, α4β1 and α4β7 integrin species and that a difference in the mode is also found such that α4β1 binds to both OPN not yet cleaved with thrombin (non-cleavage-type OPN) and the N-terminal fragment of thrombin-cleaved OPN (cleavage-type OPN), while α9β1 binds only to the thrombin-cleavage-type OPN. (Y. Yokosaki et al., (1999): The Journal of Biological Chemistry 274, 36328–36334/P. M. Green et al., (2001): FEBS Letters 503, 75–79/S. T. Barry et al., (2000): Experimental Cell Research 258, 342–351). These integrin subunits α9 and α4 or the integrin subunits β1 and β7 are highly similar in terms of amino acid sequence to each other. Additionally, the integrin species α4β1 and α4β7 are mainly found in lymphocytes and monocytes, while in neutrophils, the integrin species are expressed very slightly. Alternatively, α9β1 is highly expressed selectively in neutrophils and has functions essential for neutrophil migration through VCAM-1 and Tenascin-C. Additionally, the integrin is also expressed diversely in muscular cells, epithelial cells and liver cells and the like. As described above, the cytoplasm domains of the integrin subunits α4 and α9 cooperatively promote leukocyte migration toward inflammatory sites and aggregation therein, via individual cellular signal transmission pathways subtly differing from each other, to enhance their infiltration activities. In such manner, the integrin subunits are involved in various inflammatory reactions.
As described above, various types of integrin species promote leukocyte migration and are thus involved in inflammatory reactions. Therefore, pharmaceutical substances inhibiting these integrin activities may have a potential usefulness as an anti-inflammatory agent. For example, the integrin αVβ3 is expressed in osteoclast cells, vascular endothelial cells and smooth muscle cells and the like. An anti-αVβ3 antibody is now under way of development, which will work to inhibit the binding between the integrin αVβ3 and various binding ligands thereof to potentially exert for example an action to suppress articular damages.
Because receptors of the integrin family commonly emerge in diverse tissues to provide essential functions for the control of vital activities, however, the use of antibodies against integrin for the therapeutic treatment of rheumatoid arthritis and osteoarthritis may possibly elicit the same inhibition at other sites and may also cause the occurrence of side effects.
Additionally, WO 01/71358 discloses a screening method for a substance inhibiting the binding between the α4 integrin and osteopontin and a method for therapeutically treating inflammatory diseases, using the substance recovered by such screening.
Various factors have been indicated for the pathogenesis of rheumatoid arthritis. Thus, many reports have been issued therefor. However, not any of them is reliable. Further, currently known therapeutic methods thereof are nosotropic and have not been essentially satisfactory.
Hence, it has been strongly desired to definitely elucidate the pathogenesis of rheumatoid arthritis and provide a more excellent therapeutic method thereof. It is an object of the invention to solve such problems.
Further, rheumatoid arthritis is hardly discriminated from osteoarthritis. Therefore, it is an additional object of the invention to provide a diagnostic method thereof.
The inventors found that the OPN concentration in the articular cavity fluids of rheumatism patients and osteoarthritis patients was at a higher value. Additionally, the inventors found the increase of the ratio of the N-terminal fragment of the thrombin-cleavage type in the total OPN in rheumatism patients for the first time. Thus, the inventors speculated that OPN might be deeply involved in the onset of these diseases. Then, the inventors verified the findings at experiments using OPN knockout mice.
Further, the inventors prepared antibodies individually recognizing the N-terminal fragment and the C-terminal fragment discriminatively from the thrombin-cleaved OPN. Then, the inventors found at experiments using them that the N-terminal fragment of the thrombin-cleaved OPN was at a high concentration in the articular cavity fluids of patients with rheumatoid arthritis, in particular.
Still further, the inventors focused their attention to the fact that the N-terminal fragment of high concentration is observed in the case of the patients with rheumatoid arthritis, and the fragment contains both the RGD and the SVVYGLR (residues 11–17 of SEQ ID NO: 1) sequence sites capable of being recognized by human-type integrin. Then, the inventors anticipated that an antibody capable of blocking both the sequence sites simultaneously would inhibit the binding between OPN and integrin so broadly that the antibody could be effective for the therapeutic treatment of rheumatoid arthritis and osteoarthritis.
Further, OPN is distributed in kidney, placenta, ovary, brain, skin and the like, but is mainly expressed in bone tissue. The inventors considered that for the therapeutic treatment of rheumatoid arthritis, the binding between OPN and integrin would preferably be blocked by a method more specific to the OPN side. Because the diverse integrin species might be involved in inflammation in a cooperative manner, then, the inventors considered that it would be effective to more broadly block the binding to these diverse integrin species.
Therefore, the inventors prepared an antibody which can inhibit the binding between the RGD sequence site of human OPN and integrin and also the binding between the SVVYGLR (residues 11–17 of SEQ ID NO: 1) sequence site of human OPN and integrin, and then verified the effects thereof at experiments for cell adhesion and cell migration and the like. Further, the inventors recovered an antibody against a synthetic peptide corresponding to the inner sequence of murine OPN, to examine the efficacy of such antibody as a therapeutic agent, using an arthritis-diseased model in mouse.
More specifically, because murine OPN has the sequences RGD and SLAYGLR (SEQ ID NO: 52) recognizable by murine integrin, which are located at positions homologous to human OPN in terms of amino acid sequence, an antibody M5 was recovered as an antibody simultaneously blocking these sequences. It was verified that the binding of the antibody M5 with murine OPN and the thrombin digestion products thereof was inhibited by the peptide GRGDSP (SEQ ID NO: 55) including the sequence RGD and that the antibody M5 inhibited the migration of TNF-α-activated monocyte derived from murine spleen. It was also observed that the antibody M5 had an action to suppress bone damage when examined in a murine calvaria organ culture system. Further, it was confirmed that the antibody had an apparent therapeutic effect when administered to a murine collagen arthritis model.
The aforementioned results strongly suggest that the antibody, which can simultaneously block the binding of the RGD and SVVYGLR (residues 11–17 of SEQ ID NO: 1) sequence sites with human-type integrin, can inhibit the binding between OPN and integrin so as to be effective for the therapeutic treatment of rheumatoid arthritis and the like. Furthermore, the results suggest that the antibody can possibly be effective not only for rheumatism such as juvenile articular rheumatism and chronic rheumatism but also for psoriatic arthritis and psoriasis. Furthermore, chronic rejections after organ transplantation are characterized by complication with vascular and bronchial occlusive disorders. The results of histological examinations thereof suggest that activation of T cell and macrophage triggers generation of cytokine and growth factors, leading to disorders of vascular endothelial cell and proliferation of vascular smooth muscle cell which may lead to, via fibrogenesis and the like, vascular occlusion (P. Freese et al., (2001): Nephrol Dial Transplant, 16, 2401–2406/J. R. Waller et al., (2001): British Journal of Surgery, 88, 1429–1441/S. R. Lehtonen et al., (2001): Transplantation, 72, 1138–1144). And it is reported that OPN has an essential function for macrophage activation and fibrogenesis of vascular smooth muscle cell (A. O'Regan et al., (2000): Int J Exp Pathol, 81, 373–390). Thus, the OPN inhibitory antibody of the invention suppresses the migration of monocyte and neutrophil, thereby possibly suppressing a process toward such fibrogenesis. Thus, the antibody suppresses chronic rejections after organ transplantation, with the resultant contributions to organ adhesion. Additionally, the antibody will be effective for the therapeutic treatment of autoimmune diseases including systemic autoimmune diseases, erythematosus, uveitis, Behcet disease, multiple myositis, skein proliferative nephritis, sarcoidosis and the like.
Based on the above mentioned findings, the inventors found an anti-osteopontin antibody, which can inhibit the binding between an integrin recognizing the site of the amino acid sequence RGD and osteopontin or a fragment thereof, and can also inhibit the binding between an integrin recognizing the site of the amino acid sequence SVVYGLR (residues 11–17 of SEQ ID NO: 1) or a corresponding sequence thereto and osteopontin or a fragment thereof. The inventors filed an international application (PCT/JP02/03382) about the antibody.